A preparation of umbilical cord mesenchymal stem cells and its application in the treatment of ovarian diseases

Abstract

The present invention relates to the field of pharmaceutical technology. Specifically, a short peptide capable of specifically binding to integrin αvβ5 is obtained by screening through phage display technology. This short peptide serves as a ligand for integrin αvβ5. Further, the short peptide is anchored on the surface of human umbilical cord mesenchymal stem cells by means of click chemistry, and then a preparation of umbilical cord mesenchymal stem cells is prepared for precise targeted treatment of ovarian diseases.

Description

Technical Field

[0001] The present invention relates to a preparation containing umbilical cord mesenchymal stem cells, and particularly to an umbilical cord mesenchymal stem cell preparation for treating ovarian diseases, and a preparation method and application thereof. Background Art

[0002] Ovarian cancer (OC) is the most common gynecological tumor. Due to the insidious onset, poor prognosis and high mortality rate of OC, early diagnosis and treatment are of great significance for OC. Mesenchymal stem cells can secrete a large number of cytokines and exosomes, etc. Among them, exosomes are extracellular vesicles with a diameter between 30-100 nm. They are spherical particles enclosed by a phospholipid bilayer released by different types of cells, and contain various proteins, lipids and genetic materials, etc. These inclusions can be transferred from one cell to another cell and play a role in the target cell. In recent years, studies have found that exosomes can be used as biomarkers for OC and drug carriers for OC immunotherapy. Therefore, exosomes are inhibitors of OC progression, and they can play an anti-OC role as ideal drug carriers and therapeutic targets.

[0003] However, the targeting specificity of exosomes secreted by umbilical cord mesenchymal stem cells is poor, and its application is severely restricted. Therefore, the targeting of umbilical cord mesenchymal stem cells is an urgent problem to be solved.

[0004] Integrin is an adhesion factor on the cell surface. It participates in various physiological activities of cells by mediating the interaction between cells and between cells and the extracellular matrix. Integrin is closely related to the occurrence, development, invasion and metastasis of tumors. Existing studies have shown that integrin αvβ5 is highly expressed in ovarian cancer and can be used as a biomarker for ovarian cancer, providing a new idea for the treatment of ovarian cancer. Summary of the Invention

[0005] In order to improve the targeting problem of umbilical cord mesenchymal stem cells, the present invention uses integrin αvβ5 highly expressed in ovarian cancer as a target to guide the binding of umbilical cord mesenchymal stem cells to ovarian cancer cells, and finally treats ovarian cancer through umbilical cord mesenchymal stem cells.

[0006] One aspect of the present invention is to provide a short peptide that specifically binds to integrin αvβ5. Preferably, the short peptide of the present invention is obtained by screening through phage display technology. Specifically, the present invention uses phage display technology to screen a peptide segment with high binding specificity to integrin αvβ5 from a polypeptide library. Specifically, the amino acid sequence of the short peptide of the present invention is shown as SEQ ID No.1: RGDLGRPA.

[0007] This short peptide can act as an integrin ligand, bind to the corresponding integrin receptor on the ovarian cancer cell membrane, and anchor umbilical cord mesenchymal stem cells to ovarian cancer cells.

[0008] Another aspect of the present invention is to provide a kind of mesenchymal stem cells derived from human umbilical cord blood, and its preparation method is as follows: (1) Select human umbilical cord blood stem cells and conduct routine culture in a serum-free manner; (2) Conduct culture by passage, and passage to the 5th generation to amplify a large number of human umbilical cord mesenchymal stem cells.

[0009] Another aspect of the present invention is to provide a preparation of mesenchymal stem cells derived from human umbilical cord blood, and the preparation contains umbilical cord mesenchymal stem cells.

[0010] Furthermore, the short peptide with high binding specificity is anchored to the surface of umbilical cord mesenchymal stem cells by click chemistry.

[0011] Furthermore, the click chemistry method is to introduce complementary click chemistry functional groups on the short peptide and the surface of umbilical cord mesenchymal stem cells respectively. The click chemistry functional groups are azide compounds and alkynes, and the short peptide is anchored to the surface of umbilical cord mesenchymal stem cells through click chemical reactions.

[0012] Furthermore, the short peptide with high binding specificity of the present invention specifically binds to integrin αvβ5 highly expressed on the surface of ovarian cancer cells through ligand binding, and guides umbilical cord mesenchymal stem cells to the surface of ovarian cancer cells to achieve targeted therapy.

[0013] Furthermore, the preparation method of the preparation includes the following steps:

[0014] Step (1): Screen a short peptide that specifically binds to integrin αvβ5 through phage display technology;

[0015] Step (2): Isolate mesenchymal stem cells from human umbilical cord blood for routine culture and passage. After passage to the 5th generation, amplify and culture to harvest a large number of umbilical cord mesenchymal stem cells;

[0016] Step (3): Anchor the short peptide in step (1) to the surface of the umbilical cord mesenchymal stem cells in step (2) by click chemistry to prepare a preparation of umbilical cord mesenchymal stem cells.

[0017] Furthermore, the click chemistry method is to introduce complementary click chemistry functional groups on the short peptide and the surface of umbilical cord mesenchymal stem cells respectively. The click chemistry functional groups are azide compounds and alkynes, and the short peptide is anchored to the surface of umbilical cord mesenchymal stem cells through click chemical reactions.

[0018] Another aspect of the present invention is to provide a drug for treating ovarian cancer, the drug comprising human umbilical cord mesenchymal stem cells, wherein a short peptide with high specific binding to integrin αvβ5 is anchored on the surface of the umbilical cord mesenchymal stem cells, and the amino acid sequence of the short peptide is as shown in SEQ ID No.1.

[0019] Another aspect of the present invention is to provide an application of a preparation in the treatment of ovarian cancer, the preparation comprising human umbilical cord mesenchymal stem cells, wherein a short peptide with high specific binding to integrin αvβ5 is anchored on the surface of the umbilical cord mesenchymal stem cells, and the amino acid sequence of the short peptide is as shown in SEQ ID No.1.

[0020] Another aspect of the present invention is to provide an application of a drug in the treatment of ovarian cancer, the drug comprising human umbilical cord mesenchymal stem cells, wherein a short peptide with high specific binding to integrin αvβ5 is anchored on the surface of the umbilical cord mesenchymal stem cells, and the amino acid sequence of the short peptide is as shown in SEQ ID No.1.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) The present invention screens and obtains a short peptide that only binds to integrin αvβ5 through phage display technology, showing the characteristics of high binding specificity, providing a basis for the precise targeted treatment of subsequent umbilical cord mesenchymal stem cells.

[0023] (2) The short peptide of the present invention is anchored on the surface of umbilical cord mesenchymal stem cells by click chemistry. The principle is to use the high specific binding of the short peptide to αvβ5 highly expressed on the surface of ovarian cancer cells to guide the umbilical cord mesenchymal stem cells to the surface of ovarian cancer cells, achieving precise targeted treatment.

[0024] (3) Compared with the prior art, the preparation of umbilical cord mesenchymal stem cells containing the short peptide of the present invention can achieve precise targeted treatment of ovarian cancer cells, effectively kill ovarian cancer cells in in vitro cell experiments, and effectively reduce the lesion area and inhibit the growth of ovarian tumors in in vivo animal experiments. Description of the Drawings

[0025] Figure 1 In vivo animal experiment - tumor volume measurement results;

[0026] Figure 2 In vivo animal experiment - WB map of CA125 protein. Detailed Embodiments

[0027] The present invention will be further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0028] Example 1 Preparation of a short peptide that binds to integrin αvβ5 with high specificity

[0029] (1) Library construction and amplification

[0030] A series of random DNA sequences were designed and cloned into the coding region of the coat protein of bacteriophage M13 to construct an 8-peptide library;

[0031] (2) Target capture

[0032] The integrin αvβ5 molecule highly expressed on the surface of ovarian cancer cells is fixed on the surface of a solid phase carrier, and the library in step (1) is incubated with the solid phase carrier for 4 hours to allow the two to bind;

[0033] (3) Washing

[0034] Repeated washing to remove unbound nonspecific phages to remove unbound and non-target specific phages;

[0035] (4) Elution

[0036] After a brief incubation with a low pH buffer or by competitive elution, the target-bound phage is isolated;

[0037] (5) Infection stage

[0038] The eluted phages are used to infect bacteria to amplify the selected phages, thereby forming a new phage library for the next round of biopanning.

[0039] After five rounds of bio-panning, a short peptide with high binding specificity was screened out. After sequencing, the amino acid sequence of the short peptide was shown as SEQ ID No. 1.

[0040] Example 2 Preparation of human umbilical cord mesenchymal stem cell preparation

[0041] Human umbilical cord tissue was obtained from a healthy, full-term fetus delivered vaginally and stored in a sterile PBS solution containing 1% double-antibody. Before use, the umbilical cord was cleaned with saline and transferred to a new culture medium. It was then soaked in 75% medical alcohol for 2 minutes. After disinfection, the cord was placed in a culture dish containing saline, washed 2-3 times, and the serum was removed. First, the umbilical cord was unfolded with two forceps, and two veins and one artery were removed. The cord was then minced and evenly divided into T75 culture flasks for culture. 4 ml of complete culture medium was added to each flask. The cell flask was placed flat so that the tissue was as evenly distributed as possible across the entire bottom surface. The cells were then cultured in an incubator at 37°C and 5% CO2. On the 5th day after primary culture preparation, the medium was fully exchanged every 3 days. When the cell confluence reached 85%-90%, the cells were passaged until the 5th generation. The 5th generation was expanded and a large number of umbilical cord mesenchymal stem cells were harvested.

[0042] The short peptide of Example 1 and the above-mentioned umbilical cord mesenchymal stem cells were used to anchor the short peptide to the surface of umbilical cord mesenchymal stem cells by click chemistry. The specific steps were to introduce an azide functional group on the surface of the short peptide to prepare a compound, introduce an alkyne group on the surface of umbilical cord mesenchymal stem cells, and complete the reaction under copper catalysis to anchor the short peptide to the surface of umbilical cord mesenchymal stem cells, thereby preparing a preparation of umbilical cord mesenchymal stem cells containing the short peptide.

[0043] Example 3 Application of the preparation of Example 2 in the treatment of ovarian cancer

[0044] 1. In vitro experiment

[0045] The ovarian cancer cell line SK-OV-3 was cultured routinely and passaged to a 96-well plate for further culture. It was divided into 4 groups, namely the normal culture group, the PBS treatment group, the conventional umbilical cord mesenchymal stem cell treatment group, and the umbilical cord mesenchymal stem cell treatment group containing the short peptide. After culturing for 3 days until the cell density reached 80%, the medium was changed. The normal culture group was not treated, the PBS treatment group was added with 100 μl of PBS, the conventional umbilical cord mesenchymal stem cell treatment group was added with 100 μl of umbilical cord mesenchymal stem cells, and the umbilical cord mesenchymal stem cell treatment group containing the short peptide was added with 100 μl of the umbilical cord mesenchymal stem cells containing the short peptide of Example 2. After incubating for 24 h in each group, the cells were washed with PBS, and the cell viability was detected by the MTT method.

[0046] The detection results showed that both the umbilical cord mesenchymal stem cell treatment group containing the short peptide and the conventional umbilical cord mesenchymal stem cell treatment group had a significant effect on inhibiting the proliferation of tumor cells, and the umbilical cord mesenchymal stem cell treatment group containing the short peptide had a better inhibitory effect compared with the conventional umbilical cord mesenchymal stem cell treatment group. There was no difference between the normal culture group and the PBS treatment group, and they had no inhibitory effect on tumors (Table 1).

[0047] Table 1

[0048]

[0049] 2. In vivo experiment

[0050] SK-OV-3 ovarian cancer cells were injected into the lower axilla of the hindlimbs of rats to establish an animal model. Subsequently, rats with similar tumor volumes were randomly divided into four groups: a non-treatment group, a PBS treatment group, a conventional umbilical cord mesenchymal stem cell treatment group, and a short peptide-containing umbilical cord mesenchymal stem cell treatment group. The non-treatment group received no treatment. The PBS treatment group was injected with PBS via the tail vein. The conventional umbilical cord mesenchymal stem cell treatment group was injected with conventionally extracted umbilical cord mesenchymal stem cells via the tail vein. The short peptide-containing umbilical cord mesenchymal stem cell treatment group was injected with the short peptide-containing umbilical cord mesenchymal stem cells of Example 2 via the tail vein. Each group was given drug intervention 14 days after the establishment of the animal model. On the 21st day after drug intervention, the tumors of each group of rats were removed for volume detection, and proteins were extracted for WB quantitative analysis of ovarian cancer marker proteins.

[0051] The results showed that the short peptide-containing umbilical cord mesenchymal stem cell treatment group had a significant inhibitory effect on ovarian tumors in rats. Followed by the conventionally extracted umbilical cord mesenchymal stem cell treatment group, there was almost no difference in tumor volume between the PBS treatment group and the non-treatment group ( Figure 1 ). WB analysis of the ovarian cancer marker protein CA125 showed results consistent with the tumor inhibitory effect observed by the naked eye. The experimental results indicated that the short peptide-anchored umbilical cord mesenchymal stem cells enabled the umbilical cord mesenchymal stem cells to accurately target ovarian cancer tissues, achieving precise targeted therapy and effectively improving the therapeutic effect of umbilical cord mesenchymal stem cells ( Figure 2 ).

[0052] The basic principles, main features, and advantages of the present invention have been described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A short peptide that specifically binds to integrin αvβ5, characterized in that, The amino acid sequence of the short peptide is shown as SEQ ID No.

1.

2. A preparation of umbilical cord mesenchymal stem cells, characterized in that, The preparation contains umbilical cord mesenchymal stem cells, and the short peptide specifically binding to integrin αvβ5 as claimed in claim 1 is anchored on the surface of the umbilical cord mesenchymal stem cells.

3. The preparation according to claim 2, characterized in that, The short peptide is anchored on the surface of umbilical cord mesenchymal stem cells by click chemistry.

4. The preparation according to claim 3, characterized in that, The click chemistry method is to introduce complementary click chemistry functional groups on the surface of the short peptide and umbilical cord mesenchymal stem cells respectively. The click chemistry functional groups are azide compounds and alkynyl groups, and the short peptide is anchored on the surface of umbilical cord mesenchymal stem cells through click chemical reaction.

5. The preparation method of the preparation according to claim 2, characterized in that, The method comprises the following steps: Step (1): Screening and obtaining a short peptide specifically binding to integrin αvβ5 by phage display technology; Step (2): Culturing and amplifying umbilical cord mesenchymal stem cells; Step (3): Anchoring the short peptide in step (1) on the surface of the umbilical cord mesenchymal stem cells in step (2) by click chemistry to prepare an umbilical cord mesenchymal stem cell preparation.

6. The method according to claim 5, characterized in that, The click chemistry method is to introduce complementary click chemistry functional groups on the surface of the short peptide and umbilical cord mesenchymal stem cells respectively. The click chemistry functional groups are azide compounds and alkynyl groups, and the short peptide is anchored on the surface of umbilical cord mesenchymal stem cells through click chemical reaction.

7. Use of the preparation according to claim 2, characterized in that, The application is for preparing a drug for treating ovarian diseases. Specifically, the ovarian disease is ovarian cancer.

8. The application according to claim 7, wherein The treatment is achieved by the specific binding of the short peptide to integrin αvβ5 expressed on the surface of ovarian cancer cells, and then targeting the umbilical cord mesenchymal stem cells to ovarian cancer cells through the short peptide, so as to achieve the effect of targeted treatment of ovarian cancer.

9. A drug for treating ovarian diseases, characterized in that, The drug contains the preparation as claimed in claim 2.

10. The medicament according to claim 9, characterized in that, The drug further contains a pharmaceutically acceptable carrier.

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